U.S. patent number 4,153,417 [Application Number 05/818,646] was granted by the patent office on 1979-05-08 for method of indicating rheumatoid factors.
This patent grant is currently assigned to Pharmacia Diagnostics AB. Invention is credited to Henning R. Hallgren, Leif E. Wide.
United States Patent |
4,153,417 |
Hallgren , et al. |
May 8, 1979 |
Method of indicating rheumatoid factors
Abstract
A method of indicating rheumatoid factors belonging to at least
one of the immunoglobulin classes IgM, IgG and IgA in an aqueous
sample is disclosed. According to this method any complement factor
C1q present in the sample is pacified in a manner known per se,
whereafter the sample is reacted with soluble, aggregated
immunoglobulin labelled with one or more analytically indictable
atoms or groups to form aggregates between rheumatoid factors and
the aggregated, labelled immunoglobulin, said aggregate being
precipitated out, whereafter the precipitate is separated and the
analytically indicatable atoms or groups are indicated in the
precipitation phase and/or in the solution.
Inventors: |
Hallgren; Henning R.
(Upplands-Balinge, SE), Wide; Leif E. (Uppsala,
SE) |
Assignee: |
Pharmacia Diagnostics AB
(Uppsala, SE)
|
Family
ID: |
20328828 |
Appl.
No.: |
05/818,646 |
Filed: |
July 25, 1977 |
Foreign Application Priority Data
Current U.S.
Class: |
435/7.92;
435/962; 436/509; 436/513; 436/528; 436/536; 436/537; 436/545;
436/546; 436/800; 436/804; 436/819; 436/821; 436/826 |
Current CPC
Class: |
G01N
33/564 (20130101); Y10S 435/962 (20130101); Y10S
436/80 (20130101); Y10S 436/819 (20130101); Y10S
436/826 (20130101); Y10S 436/821 (20130101); Y10S
436/804 (20130101) |
Current International
Class: |
G01N
33/564 (20060101); G01N 033/16 () |
Field of
Search: |
;23/23B,230.6
;424/8,12,1 ;195/13.5A |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts, 71:58955x (1969). .
Chemical Abstracts, 74:2303c (1971)..
|
Primary Examiner: Marantz; Sidney
Attorney, Agent or Firm: Philpitt; Fred C.
Claims
What is claimed is:
1. A method of indicating rheumatoid factors belonging to at least
one of the immunoglobulin classes IgM, IgG and IgA in an aqueous
sample, which method comprises
(a) pacifying any complement factor C1q present in the sample,
(b) thereafter reacting the sample with soluble, aggregated
immunoglobulin labelled with one or more analytically indicatable
atoms or groups to form aggregates between rheumatoid factors and
the aggregated, labelled immunoglobulin, said aggregates being
precipitated out,
(c) separating the precipitate, and
(d) thereafter measuring indicatable atoms or groups in the
precipitated phase formed and/or in the solution.
2. A method according to claim 1 wherein the labelled aggregated
immunoglobulin belongs to the IgG class.
Description
The present invention relates to a method of indicating rheumatoid
factors belonging at least to one of the immunoglobulin classes
IgM, IgG and IgA in an aqueous sample.
Anti-immunoglobulins are also designated rheumatoid factors and may
belong to the immunoglobulin classes IgM, IgG, IgA or, possibly,
also to other immunoglobulin classes. In turn, rheumatoid factors
may be directed against immunoglobulins belonging to the classes
IgG or IgM or possibly against other immunoglobulin classes, which
immunoglobulins have been changed in structure due to immune
complex formation or aggregation.
Previously suggested test methods for indicating rheumatoid factors
are based on the agglutination of, for example, blood corpuscles or
latex particles coated with IgG. These methods indicate primarily
rheumatoid factors of the IgM-type directed against changed IgG.
Samples taken from most patients suffering from rheumatoid
arthritis show a positive result in such a test, although samples
from 20-30% show a negative result.
In accordance with the present invention there is now provided a
method of indicating rheumatoid factors in an aqueous sample, said
method indicating all rheumatoid factors in the sample in a manner
which is more complete than was possible with the previously known
methods, i.e. also rheumatoid factors which do not belong to the
immunoglobulin class IgM and which could not be previously
indicated to the desired extent in the presence or the absence of
such factors belonging to the IgM class.
The method according to the invention is characterised by the fact
that any complement factor C1q present in the sample is pacified in
a manner known per se, whereafter the sample is reacted with
soluble, aggregated immunoglobulin labelled with one or more
analytically indicatable atoms or groups to form aggregates between
rheumatoid factors and the aggregated, labelled immunoglobulin,
which aggregates are precipitated out, whereafter the precipitate
is separated and the analytically indicatable atoms or groups are
indicated in the precipitation phase and/or in the solution.
The complement factor C1q in the sample may, for example, be
pacified by heating the sample or by adding thereto diaminobutane
or deoxyribonuecleic acid in a manner known per se.
Soluble, aggregated immunoglobulin can be prepared, for example, by
heating a solution of an immunoglobulin or by chemical treatment
with bis-diazotized benzidine or di-(4-aminophenyl)-sulphone (cf.
Handbook of Experimental Immunology, Second Ed., Edited by D.M.
Weir, Blackwell Scientific Publications, Oxford, 1976, page 10.75)
and subsequently separating soluble, aggregated immunoglobulin from
monomeric immunoglobulin and from any minor quantities of insoluble
aggregates formed, by gel filtration. Preferably, the
immunoglobulin used in this context is belonging to the IgG-class.
The immunoglobulin is not aggregated more than that the major
portion of the aggregated immunoglobulin is still soluble in the
aqueous sample.
For labelling the aggregated immunoglobulin, there can be use any
analytically indicatable atom or group known with regard to the
labelling of immunoglobulins. Thus, labelling of aggregated
immunoglobulins with a radioactive isotope can be effected in a
conventional manner, there being selected for this purpose a
suitable isotope, such as .sup.125 I (see for example the method
according to Hunter and Greenwood, Nature, volume 194, 1962, page
495). Similarly, labelling can be effected with a fluorescent group
in a conventional manner, for example with the aid of a fluorescein
derivative, such as fluorescein isothiocyanate. Labelling may also
be effected with an enzymatically active group or with groups
containing free radicals for indicating purposes.
In order to obtain a more complete precipitation of the obtained
aggregates between rheumatoid factors and the aggregated labelled
immunoglobulin, methods known per se in connection with the
precipitation of macromolecules can be made use of. For instance,
there may be added a water-soluble, uncharged polymer of the type
which can be used to facilitate precipitation of macromolecules
(e.g. in connection with immunological reactions for facilitating
the precipitation of antibody-antigen complexes) by reducing the
liquid volume in the solution accessible to the macromolecules by
so-called steric exclusion, thereby to reduce the solubility of the
macromolecules (see for example Hellsing, Acta Chem. Scand. 20
(1966) page 1251). Examples of such polymers include water-soluble
polyethylene glycols, polysaccharides and (uncharged)
polysaccharide derivatives, e.g. dextran and water-soluble
cellulose derivatives. Although the polymer is preferably added
before the reaction takes place it may also be added during or
after the reaction process. The amount of polymer added is selected
in all cases in a manner such that the polymer concentration lies
immediately beneath that at which precipitation of any of the
individual components taking part in the reaction (i.e. primarily
aggregated, labelled immunoglobulin) is obtained. Suitable
concentrations can be readily established by simple tests.
The invention will now be described with reference to a specific
example.
EXAMPLE
A. Preparation of aggregated human-IgG (agg IgG)
Human-IgG (fraction II from Cohn-fractionation) from combined human
sera was obtained from Kabi AB, Sweden and was heated in the form
of a 2% IgG-solution for 20 minutes at 60.degree. C. The thus
obtained aggregated IgG (agg IgG) was separated from monomeric IgG
by gel-filtration on a 90.times. 1.5 cm column containing particles
of dextran cross-linked with epichlorohydrin (Sephadex.sup.(R)
G-200 from Pharmacia Fine Chemicals AB, Sweden) and equilibrated
with 0.1 M tris(hydroxymethyl)-aminomethane-HCl-buffer containing
0.5 M NaCl having a pH 7.4. Concentrations of agg IgG were
determined spectrophotometrically at 280 nm.
B. Preparation of labelled agg IgG
To 20 .mu.l of a solution containing 40 .mu.g agg IgG obtained
according to A above were added 500 .mu.Ci Na .sup.125 I and 10
.mu.l of 0.5 M sodium phosphate buffer having a pH 7.4 and 10 .mu.g
of chloramine T in 10 .mu.l water. After 50 seconds, 24 .mu.g of
sodium methabisulphite were added. The reaction mixture was
separated on Sephadex.sup.(R) G-200 (i.e. gel particles consisting
of dextran cross-linked with epichlorohydrin), the first fraction
with the void volume being recovered. The eluted, labelled agg IgG
was centrifuged at 3,500 g for 5 minutes to remove spontaneously
precipitatable IgG. The labelled protein was diluted to
approximately 40 .mu.g/l (40,000 cpm in 0.1 ml) with a buffer
solution prepared from 500 ml of 0.1 M sodium phosphate buffer
having a pH 7.5, 500 ml of 0.15 M NaCl, 10 ml of 5% (w/v) NaN.sub.3
and 5 ml of Tween.sup.(R) 20 (i.e. polyoxyethylene (20) sorbitan
monolaurate).
C. Determination of rheumatoid factor-activity
Blood samples were taken aseptically from patients and permitted to
clot at room temperature, whereafter they were centrifuged at 3,000
g and serum recovered. The serum was heat treated for 30 minutes at
a temperature of 56.degree. C.
The serum was then diluted to 1:40 with a solution having the
following composition: 500 ml of 0.1 M sodium phosphate buffer pH
7.5, 500 ml of 0.15 M NaCl, 10 ml of 5% NaN.sub.3, 5 ml of
Tween.sup.(R) 20 and 2 g of polyethylene glycol (molecular weight
6,000). 400 .mu.l of said serum dilution and 100 .mu.l of I.sup.125
labelled agg IgG (40,000 cpm) (obtained according to B) were
charged to plastic tubes. The tubes were plugged and the contents
incubated under constant rotation for 16 hours at +4.degree. C.
Thereafter the contents of the tubes were centrifuged at 3,500 g
for 3 minutes. The plastic plugs were removed and 2 ml of a 0.9 M
NaCl solution containing 0.5% Tween.sup.(R) 20 were added to each
tube. The contents of the tubes were centrifuged at 3,500 g for 3
minutes. The supernatant was removed by suction. This washing
procedure was repeated three times. The tubes were then plugged and
placed in an automatic gamma counter.
High measurement values were obtained with samples taken from
patients suffering from rheumatoid arthritis. Compared with
conventional measuring techniques, better agreement was obtained
between the measuring results and clinical diagnosis when using the
present method.
28 patients suffering with joint complaints, suspected to be some
form of rheumatoid arthritis, and which had shown negative results
when examined according to conventional methods, were examined by
the method according to the invention whereupon elevated
measurement values indicating the presence of rheumatoid factors
were obtained in 16 cases.
* * * * *